Blood coagulation is both accomplished and controlled by highly-specific proteolytic enzymes. Most function as part of a membrane-bound multicomponent complex, associated with a non-enzymatic protein cofactor that is required for activity in vivo. Although the enzymes are homologous, the cofactors in the complexes include both soluble and integral membrane proteins, and are very diverse in sequence and size. The primary goal of our studies is to elucidate the molecular mechanisms by which the cofactors control enzyme activity. During the current grant period, our unique approach to investigating these complexes has shown that the active sites of these complexes are located far (greater than 60 Angstroms) above the membrane surface, and has identified two types of cofactor-dependent structural changes that coincide with the stimulation of enzyme activity: changes in the conformation of the enzyme active site and changes in active site location or orientation relative to the membrane surface. We have further found that in one case the cofactor actually elicited two separate conformational changes in the enzyme active site, but only one of them correlated with the cofactor-dependent activation of the substrate. The experiments proposed here will identify for each membrane-bound complex which cofactor-dependent changes in the conformation of the active site are functionally important, and will determine whether the height or orientation of the active site above the membrane surface is an important mechanism for regulating enzyme cleavage of the membrane-bound substrates. A comparison of the results will indicate whether or not the structurally diverse cofactors utilize the same molecular mechanisms to regulate enzyme activity. This will be accomplished by positioning a fluorophore at different locations in the active site groove of an enzyme and monitoring its sensitivity to the binding of the intact cofactor and of various functional and non-functional domains of the cofactor. This will indicate the regions of the active site that experience a cofactor-dependent change in conformation, the domain of the cofactor that elicits each conformational change, and the conformational changes that correlate with function. Singlet-singlet energy transfer will be used to measure the distance between acceptor dyes at the phospholipid surface and the donor dye in the active site of an enzyme in the presence of either inactivated cofactor or individual domains of the cofactor to determine whether active site location correlates with function and which cofactor domains mediate the movement of the enzyme active site. Thrombin binds to thrombomodulin (TM), an integral membrane glycoprotein cofactor, far above the membrane surface. To identify the structural components of TM required to maintain the thrombin active site at 66 Angstroms above the membrane surface, the effect of removing the carbohydrate moieties and/or the N-terminal domain of TM on this height will be measured using fluorescence energy transfer.